Vesicle capturing devices and methods for using same

ABSTRACT

Provided is a device that collects vesicles and vesicle-like materials from biological fluids. Such devices comprise at least one sample loading region; at least one corresponding vesicle-capture material, wherein said vesicle-capture material comprises glass-like materials; and at least one corresponding sample receiving region, wherein passage of the biological fluid from the sample loading region through the vesicle capture material and into the sample receiving region results in the capture of vesicles. Additional methods provide for a method of isolating vesicles and vesicle-like materials from biological fluids are also provided.

BACKGROUND

1. Field of the Invention

The present disclosure relates to compositions, devices, and methods forcapture of exosomes, vesicles, and other circulating membrane boundnucleic acid and/or protein-containing structures that are released fromcells into biological fluids.

2. Description of Related Art

Currently, many diagnostic tests are performed on a biological fluidsample (e.g., blood, urine, etc.) extracted from a patient for thediagnosis or prognosis of disease. The diagnosis or prognosis may bederived from identification of a biomarker or a biochemical pattern thatis not present in healthy patients or is altered from a previouslyobtained patient sample. However, these diagnostic tests are typicallybased upon the presence of known and well characterized biomarkers inthe fluid sample (e.g., electrolytes, urea, creatinine, glucose, plasmaproteins such as albumins, immunoglobulins and the like, biologicalcompounds such as thiamin, riboflavin, niacin, vitamin B6, folic acid,vitamin D, biotin, or iron). Some diagnostic tests are directed todetection of specific biomarkers (e.g., cell surface proteins) that areunique to diseased cells. Some diagnostic tests are designed to detector identify disease states through the isolation and amplification ofnucleic acids, in order to study expression levels of certaindisease-associated genes.

Often, use of bodily fluids to isolate or detect biomarkerssignificantly dilutes a biomarker and results in readouts that lack therequisite sensitivity. Additionally, most biomarkers are produced in lowor even moderate amounts in tissues other than the diseased tissue, suchas normal tissues. Thus, there exists a need for improved sensitivityand accuracy in diagnostic assays that employ biological fluids such asbodily fluids.

SUMMARY

Given the need for diagnostic assays that employ biological fluids, andin particular those that exploit certain biomarkers within such fluidsthat are vesicle bound, there is provided, in several embodiments, amethod of isolating vesicles from biological fluid, comprising obtaininga biological sample comprising the vesicles, loading at least a portionof the biological sample into a sample loading region of a vesiclecapture device, passing the biological fluid sample from the sampleloading region through a vesicle-capture material in the vesicle capturedevice, the vesicle-capture material comprising glass-like materials toproduce a supernatant; and passing the supernatant to a sample receivingregion of the vesicle capture device and discarding the supernatant,wherein the passings results in capture of the vesicles within thebiological fluid on or in the vesicle-capture material.

In several embodiments, the vesicle-capture material comprisesglass-like materials. In several embodiments the vesicle-capturematerial comprises a plurality of layers of the material. In someembodiments, the retention rate of the vesicle-capture material isgreater than 50%, 75%, 90% or 99% for vesicles having a diameter of fromabout 0.6 microns to about 1.5 microns in diameter. In one embodiment,the vesicle-capture material captures vesicles sized from about 0.7microns to about 1.6 microns in diameter. In one embodiment, thevesicle-capture material captures exosomes or other vesicles ranging insize from about 0.020 to about 1.0 microns.

In several embodiments, combinations of vesicle capture materials areused. In some embodiments, a plurality of glass-like materials are used.In several embodiments, a plurality of layers of materials are used. Inseveral embodiments, the plurality of layers of the vesicle-capturematerial comprises at least a first layer and a second layer ofglassfiber. In some embodiments, the biological fluid is passed throughthe first layer of glassfiber so as to capture material from thebiological sample that is about 1.6 microns or greater in diameter. Insome embodiments, the biological fluid is passed through the secondlayer of glassfiber so as to capture vesicles having a minimum size fromabout 0.6 microns to about 0.8 microns in diameter, and having a maximumsize of less than 1.6 microns. In several embodiments, combinations ofglass-like and non-glass-like materials are used. In one embodiment, anon glass-like material comprising nitrocellulose is used in combinationwith a glass-like material.

In several embodiments, the vesicle-capture material is modified inorder to tailor the profile of vesicles that are captured. In oneembodiment, the zeta potential of the material is used as a basis formodification (e.g., electrostatic charging) of the material. In severalembodiments, the material (based on its zeta potential) does not requiremodification.

In several embodiments, the methods disclosed herein further compriseeluting the vesicles from the vesicle-capture material. As such, in someembodiments, the vesicle-capture material is optimized to balance theattractive nature of the material and the ability of the material torelease captured vesicles.

In several embodiments, the vesicle-capture device can be connected to avacuum source in order to pass the biological fluid from the sampleloading region through the vesicle-capture material and into the samplereceiving region. In one embodiment, the passings are accomplishedthrough the application of vacuum pressure to the device. In severalembodiments, the vesicle-capture device can receive positive pressure inorder to pass the biological fluid from the sample loading regionthrough the vesicle-capture material and into the sample receivingregion. In one embodiment, the passings are accomplished through theapplication of positive pressure to the device. In several embodiments,the device can be placed in a centrifuge in order to pass the biologicalfluid from the sample loading region through the vesicle-capturematerial and into the sample receiving region. In one embodiment, thepassings are accomplished through low-speed centrifugation of thedevice. In several embodiments, the vesicle capture device is configuredin a multi-well plate format.

There is also provided herein a method for isolating a biomarker,comprising isolating vesicles comprising at least one biomarker from abiological fluid by passing the biological fluid through avesicle-capture material, removing non-vesicle material from thevesicle-capture material and lysing the vesicles in or on thevesicle-capture material with a lysis buffer, thereby isolating abiomarker from the vesicles.

In some embodiments, the biomarker is selected from the group consistingof RNA, DNA, protein, and carbohydrate. In several embodiments, the RNAis of a type selected from the group consisting of mRNA, miRNA, rRNA,tRNA, and vRNA.

There is also provided, in several embodiments, a vesicle capture devicecomprising a biological fluid sample loading region, a vesicle-capturematerial comprising at least a first and a second layer of glassfiber,wherein the first layer is closer to the sample loading region than thesecond layer, wherein the first layer of glassfiber captures materialfrom the biological sample that is about 1.6 microns or greater indiameter, wherein the second layer of glassfiber captures vesicleshaving a minimum size from about 0.6 microns to about 0.8 microns indiameter, and having a maximum size of less than 1.6 microns; and abiological fluid sample receiving region, wherein passing of thebiological fluid sample from the sample loading region to the samplereceiving region results in capture of vesicles within the biologicalfluid sample on or in the vesicle-capture material.

In one embodiment, the vesicle-capture device is configured to beconnected to a vacuum source in order to pass the biological fluidsample from the sample loading region through the vesicle-capturematerial and into the sample receiving region. In one embodiment, thevesicle-capture device is configured to receive positive pressure inorder to pass the biological fluid sample from the sample loading regionthrough the vesicle-capture material and into the sample receivingregion. In one embodiment, the device is configured to be placed in acentrifuge in order to pass the biological fluid sample from the sampleloading region through the vesicle-capture material and into the samplereceiving region.

Some embodiments provide a device for the collection of vesicles from abiological fluid, the device comprising (1) at least one sample loadingregion; (2) at least one corresponding vesicle-capture material and (3)at least one corresponding sample receiving region, wherein passage ofthe biological fluid from the sample loading region through thevesicle-capture material and into the sample receiving region results incapture of vesicles within the biological fluid on or in thevesicle-capture material. In some embodiments, wherein thevesicle-capture material comprises glass-like materials, which have astructure that is disordered or “amorphous” at the atomic scale, likeplastic or glass. Glass-like materials include, but are not limited toglass beads or fibers, silica beads (or other configuration),nitrocellulose, nylon, polyvinylidene fluoride (PVDF) or other similarpolymers, metal or nano-metal fibers, polystyrene, ethylene vinylacetate or other co-polymers, natural fibers (e.g., silk), alginatefiber, poly NZPA, or combinations thereof. In certain embodiments, thevesicle-capture material optionally comprises a plurality of layers ofvesicle-capture material. In other embodiments, the vesicle-capturematerial further comprises nitrocellulose. In some embodiments, thevesicle-capture material captures exosomes ranging in size from about 50to about 100 nanometers.

In some embodiments, the device is comprises a multi-well plate format.In other embodiments, the device is can be placed in a centrifuge inorder to pass the biological fluid from the sample loading regionthrough the vesicle-capture material and into the sample receivingregion. In some embodiments, the device is can be connected to a vacuumsource in order to pass the biological fluid from the sample loadingregion through the vesicle-capture material and into the samplereceiving region. In other embodiments, the device can receive positivepressure in order to pass the biological fluid from the sample loadingregion through the vesicle-capture material and into the samplereceiving region. In still additional embodiments, the passage of thebiological fluid into the sample receiving region is achieved by gravitypressure or in other embodiments by wicking-type materials.

Some embodiments provide a method of isolating vesicles from biologicalfluid, comprising (1) obtaining a biological sample comprising vesicles;(2) loading at least a portion of the biological sample into a sampleloading region of a vesicle capture device; (3) passing the biologicalsample from the sample loading region through a vesicle-capture materialin the vesicle capture device, the vesicle-capture material comprisingglass-like materials; and (4) passing the biological sample from thevesicle-capture material to a sample receiving region of the vesiclecapture device, wherein the passages of the biological sample results incapture of the vesicles within the biological fluid on or in thevesicle-capture material. In some embodiments, the vesicle-capturematerial further comprises nitrocellulose. In other embodiments, themethod further comprises eluting the vesicles from the vesicle-capturematerial. In some embodiments, the passing is accomplished through theapplication of vacuum pressure to the device. In other embodiments, thepassing is accomplished through low-speed centrifugation of the device.In some embodiments, the method further comprises capturing, enriching,and/or condensing vesicles comprising RNA; removing non-vesicle materialfrom the device; and lysing the vesicles in or on the vesicle-capturematerial with a lysis buffer, thereby isolating vesicle-associated RNAfrom the vesicles.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F depict screening of various filters for their exosomalcapture efficiency.

FIG. 2 compares exosome capture between glassfibers and silicaparticles.

FIG. 3 depicts an mRNA analysis of saliva exosomes captured byglassfiber filters.

FIGS. 4A-4F depict scanning electron analysis of exosomes captured froma urine sample.

FIG. 5 depicts an mRNA analysis of urine exosomes and compares theeffects of water washing.

FIGS. 6A-6C depict low magnification scanning electron analysis ofexosomes captured from a urine sample.

FIGS. 7A-7C depict a comparison of the efficiency of exosome capture onfilters as compared to established ultracentrifugation methods.

FIGS. 8A-8B depict the determination of the zeta potential ofpolystyrene.

FIGS. 9A-9B depict the determination of the zeta potential of PVDFImmoblion-P.

FIGS. 10A-10B depict the determination of the zeta potential ofglassfiber.

FIGS. 11A-11B depict the determination of the zeta potential of mylonnytran.

DETAILED DESCRIPTION

Identification of specific biomarkers including, but not limited to,DNA, RNA (such as mRNA, miRNA or microRNA, and siRNA), and proteins canprovide bio-signatures that are used for the diagnosis, prognosis, ortheranosis of a condition or disease. See, e.g., Jiang Q., Wang Y., HaoY., Juan L., Teng M., Zhang X., Li M., Wang G., Liu Y., (2009)miR2Disease: a manually curated database for microRNA deregulation inhuman disease. Nucleic Acids Res 37:D98-104, which is incorporatedherein by reference. While DNA and RNA typically are contained in theintracellular environment, these nucleic acids also existextracellularly. In some cases, DNA and/or RNA are naked (e.g., notencapsulated or associated with another structure or compound. RNAses,which degrade RNA, are known to be elevated in some disease states, forexample, in certain cancers. The extracellular environment, includingthe plasma, serum, urine, or other biological fluids is known to containsubstantial quantities of RNAses. Given this context, extracellular DNA,RNA, or other biomarkers are often considered a meaningless degradationproduct in an extracellular sample, not only because their levels maynot be representative of the true levels of the intracellular message,but also due to the instability and poor quality of the nucleic acids.

Moreover, due to the rapid rate of nucleic acid degradation in theextracellular environment, conventional understanding suggests that manytissues are unable to provide nucleic acid that would be suitable as adiagnostic target, because the nucleic acids would be degraded beforethey could be used as a template for detection. However, extracellularRNA (as well as other biomarkers disclosed herein) is often associatedwith one or more different types of membrane particles (ranging in sizefrom 50-80 nm), exosomes (ranging in size from 50-100 nm), exosome-likevesicles (ranging in size from 20-50 nm), and microvesicles (ranging insize from 100-1000 nm). Other vesicle types may also be captured,including, but not limited to nanovesicles, vesicles, dexosomes, blebs,prostasomes, microparticles, intralumenal vesicles, endosomal-likevesicles or exocytosed vehicles. As used herein, the terms “exosomes”and “vesicles” shall be given their ordinary meaning and shall also beread to include any shed membrane bound particle that is derived fromeither the plasma membrane or an internal membrane. For clarity, theterms describing various types of vesicles shall, unless expresslystated otherwise, be generally referred to as vesicles or exosomes.Exosomes can also include cell-derived structures bounded by a lipidbilayer membrane arising from both herniated evagination (blebbing)separation and sealing of portions of the plasma membrane or from theexport of any intracellular membrane-bounded vesicular structurecontaining various membrane-associated proteins of tumor origin,including surface-bound molecules derived from the host circulation thatbind selectively to the tumor-derived proteins together with moleculescontained in the exosome lumen, including but not limited totumor-derived microRNAs or intracellular proteins. Exosomes can alsoinclude membrane fragments. Circulating tumor-derived exosomes (CTEs) asreferenced herein are exosomes that are shed into circulation or bodilyfluids from tumor cells. CTEs, as with cell-of-origin specific exosomes,typically have unique biomarkers that permit their isolation from bodilyfluids in a highly specific manner. As achieved by several embodimentsdisclosed herein, selective isolation of any of such type of vesiclesallows for isolation and analysis of their RNA (such as mRNA, microRNA,and siRNA) which can be useful in diagnosis or prognosis of numerousdiseases.

Conventional methods for isolation of exosomes, or other vesicles, ofteninvolve ultracentrifugation (often multiple rounds) in order to separatethe vesicles from other matter in a biological sample.Ultracentrifugation is accomplished through the use of specialized,expensive, and potentially hazardous equipment. Indeed, the tremendousrotational kinetic energy of the rotor in an operating ultracentrifugemakes the catastrophic failure of a spinning rotor a serious concern.Stresses associated with routine use eventually cause rotors todeteriorate and careful maintenance of rotors to prevent corrosion andto detect deterioration is necessary to avoid many hazards. However,even routine maintenance is beyond the capabilities of many personstrained in the use of an ultracentrifuge. Thus, maintenance can beexpensive because of the specialized knowledge required to properlymaintain and/or carefully repair such a potentially hazardous piece oflaboratory equipment. Moreover, maintenance and needed repairs canresult in delays for the user. Consequently, ultracentrifugation issometimes an impractical or impossible technique for many laboratories.

Therefore, provided herein are devices, compositions, and methods forcapture of exosomes, vesicles, and other circulating membrane bound,nucleic acid (including, but not limited to DNA, RNA, mRNA, microRNA,and siRNA) and/or protein-containing structures that are released fromcells into biological fluids. Thus, in several embodiments the devicesand methods as disclosed herein provide several advantages overtraditional techniques for vesicle isolation, such asultracentrifugation. In some embodiments, the devices and methods allowfor the enrichment, concentration, and/or isolation of vesicles,exosomes, and/or biomarkers in samples by allowing for multiplefiltrations of samples through the same filter. Consequently, increasedamounts of material can be collected simply by applying multiple samplealiquots to a device. In some embodiments, samples can be manipulatedwhile in and/or on the device. Thus, samples can be further purifiedprior to biomarker analysis by washing with solutions known to removecontaminants. In some embodiments, samples can be manipulated before,during, and/or after application to the device. Such manipulations canincrease ultimate sample purity by removing potential contaminants. Suchmanipulations also allow the user to target different fractions of thesame sample. This is particularly advantageous because a single deviceand method allows collection of various sized exosome or vesicles,whereas traditional techniques dispose of certain cellular (and/ornon-cellular) fractions while in pursuit of a different particularfraction.

In several embodiments the devices and methods as disclosed herein areeasier to use than traditional techniques for vesicle isolation, such asultracentrifugation. Some embodiments do not require pretreatment ofsamples prior to use. Pretreatment can take many forms, including samplefractionation, precipitation of unwanted material, etc. For example,some embodiments allow for samples to be taken from donors and used“as-is” for isolation and testing of biomarkers. However, someembodiments allow a user to pretreat samples for certain reasons. Thesereasons include, but are not limited to, protocols to facilitatestorage, facilitating biomarker detection, etc. Because the devices andmethods utilize filters to capture vesicles, multiple samples can be runthrough the same filter and afford collection of all vesicles in onelocation, unlike traditional methods (e.g. ultracentrifugation) that canresult in samples being collected in multiple tubes. Such traditionalmethods require manipulation by a user beyond what is minimally requiredby the disclosed methods and devices. Increased sample manipulation canincrease the difficulty of isolating vesicles and increases thelikelihood of user error, contamination, and/or sample loss.

In some embodiments, the devices disclosed herein are particularlyefficient at processing samples. For example, the device can beconfigured in a multiwall plate format, allowing the high-throughputprocessing of samples. In some embodiments, the filter(s) employed inthe device are particularly efficient at capturing vesicles from asample. In some embodiments, the capture of the vesicles outperformsother methods. In some embodiments, the capture of vesicles is roughlyequivalent to other methods, but allows for substantially greaterrelease and/or isolation of DNA, RNA (including, but not limited to,mRNA, microRNA, and/or siRNA), or protein from the captured vesicles. Insome embodiments, the cost of processing samples is reduced as comparedto traditional methods. For example, several embodiments do not requirespecialized equipment (e.g., ultracentrifuge and associated rotors)whose startup and maintenance costs are quite high and whose operationrenders them potentially hazardous. Some embodiments of the device areparticularly advantageous because of the device's efficiency whichallows for the use of low volumes of the biological sample. However, insome embodiments, repeated applications of aliquots of the biologicalsample will allow concentration (e.g., condensation, enrichment, and thelike) of the vesicles in the sample (e.g., such embodiments areparticularly useful for high volume-low vesicle concentration samplessuch as urine). In several embodiments, such concentrations allow for anenrichment of the biomarker of interest. Moreover, the devices disclosedherein are optionally self-contained, and therefore reduce the risk ofcontamination of the samples, and the possibility of the associated lossof DNA/RNA or protein that is to be analyzed (e.g., due to RNAses orproteases).

In several embodiments, devices comprising a sample loading region, avesicle-capturing material, and a sample receiving region are provided.The devices allow a biological sample to be loaded into the sampleloading region, passed over or through the vesicle-capturing material inorder to trap, temporarily hold, or otherwise isolate the vesicles fromthe remainder of the components of the biological sample, which isreceived in the sample receiving region of the device. In someembodiments, the device comprises a single sample loading region, one ormore vesicle-capturing materials, and a single sample receiving region.In several such embodiments, the devices are provided in a single useformat (e.g., are pre-sterilized and disposable). However, in someembodiments, after capture of the vesicles and subsequent processing,the device can be cleaned and/or sterilized, and re-used. In severalembodiments, the device comprises a plurality of sample loading regions,each associated with a corresponding plurality of vesicle-capturingmaterial(s), and a corresponding plurality of sample receiving regions.In some embodiments, multi-well devices are configured with standarddimensions (e.g., those of a 96 well or 384 well plate) such that thedevice can be placed in standard laboratory equipment (e.g., a standardlow-speed plate centrifuge). In still additional embodiments, the deviceis configured to interact with a device for high-throughputquantification of mRNA such as those described in U.S. Pat. No.7,745,180, issued on Jun. 29, 2010, and is incorporated be referenceherein.

In some embodiments, the vesicle-capturing material captures desiredvesicles from a biological sample. In some embodiments, therefore, thevesicle-capturing material is selected based on the pore (or otherpassages through a vesicle-capturing material) size of the material. Insome embodiments, the vesicle-capturing material comprises a filter. Insome embodiments, the filter comprises pores. As used herein, the terms“pore” or “pores” shall be given their ordinary meaning and shall alsorefer to direct or convoluted passageways through a vesicle-capturematerial. In some embodiments, the materials that make up the filterprovide indirect passageways through the filter. For example, in someembodiments, the vesicle-capture material comprises a plurality offibers, which allow passage of certain substances through the gaps inthe fiber, but do not have pores per se.

In those embodiments wherein the vesicle capture material comprises afilter, the type of filter may vary, depending on the application. Asdiscussed above, the size (or quantity) of the vesicles to be capturedis a consideration when choosing a filter. In other embodiments, afilter is chosen for overall vesicle retention capacity. In stilladditional embodiments, a filter is chosen for its efficiency inremoving vesicles. Filters suitable for use in devices disclosed hereininclude, but are not limited to glass-like materials such as glassbeads, glassfiber, silica, and the like; nitrocellulose; nylon;polyvinylidene fluoride (PVDF) and similar polymers; metal or nano-metalfibers; polystyrene; ethylene vinyl acetate or other co-polymers;natural fibers such as silk; alginate fiber; poly NZPA; or other fibrousmaterials. In some embodiments, two or more types of fibers may be used,for example by layering the materials over one another. In someembodiments, such devices may be more efficient, as each material usedcan be selected and/or positioned to optimize its filtrationcharacteristics.

The vesicle retention rate of the vesicle-capture material is dependenton the dimensions of the vesicles to be captured. The rate is defined asthe percentage retention of a vesicle (or particle) of a given size. Forexample a retention rate of 100% for would indicate that all of theparticles of a particular size are captured within the material. In someembodiments, the retention rate of the vesicle-capture material isgreater than 50%, 75%, 90% or 99% for vesicles having a diameter of fromabout 0.6 microns to about 1.5 microns in diameter. In severalembodiments, retention rate is increased by the use of a plurality oflayers of material, and/or modification of materials. In someembodiments, certain materials are used in a particular orientation inorder to pre-filter larger cellular material or debris from a sample,which thereby further enhances the vesicle retention rate.

In some embodiments, the material is modified in order in enhance itsvesicle capturing capability or to enable capture of different types ofvesicles. In some embodiments, the material may be electrocharged (e.g.,electrostatically charged), coated with hydrophilic or hydrophobicmaterials, chemically modified, or biologically modified. For example,in some embodiments, differential capture of vesicles is made based onthe surface expression of protein markers and a complementary agent onthe capture material that identifies that marker (e.g., an antibody thatrecognizes an antigen on a particular vesicle). In some embodiments, themarkers are unique vesicle proteins or peptides. In some disease states,the markers may also comprise certain vesicle modifications, which, insome embodiments, are used to isolate particular vesicles. In suchembodiments, the capture material is configured in a manner which allowsfor specific recognition of the vesicle modification. Modification ofthe vesicles may include, but are not limited to addition of lipids,carbohydrates, and other molecules such as acylated, formylated,lipoylated, myristolylated, palmitoylated, alkylated, methylated,isoprenylated, prenylated, amidated, glycosylated, hydroxylated,iodinated, adenylated, phosphorylated, sulfated, and selenoylated,ubiquitinated. In some embodiments, the capture material is configuredto recognize vesicle markers comprising non-proteins such as lipids,carbohydrates, nucleic acids, RNA, mRNA, siRNA, microRNA, DNA, etc.

Depending on the configuration of the vesicle capturing device, variousprocedures may be used to pass a biological sample through the capturematerial. For example, in one embodiment, low speed centrifugation isused. In one embodiment, vacuum pressure is used. In one embodiment,positive pressure is used. In one embodiment, gravity is used.Combinations of these procedures may also be used. Thus, the vesiclecapturing devices and associated methods described herein are highlyversatile and suitable for use in a variety of laboratories.

The biological fluids from which vesicles can be captured using thedevices disclosed herein include a variety of bodily fluids from asubject. As used herein, a “bodily fluid” shall be given its ordinarymeaning and shall also refer to a sample of fluid isolated from anywherein the body of the subject, including but not limited to, for example,blood, plasma, serum, urine, sputum, spinal fluid, pleural fluid, nippleaspirates, lymph fluid, fluid of the respiratory, intestinal, andgenitourinary tracts, tear fluid, saliva, breast milk, fluid from thelymphatic system, semen, cerebrospinal fluid, intra-organ system fluid,ascitic fluid, tumor cyst fluid, amniotic fluid and combinationsthereof. In some embodiments, the biological sample is preferablycollected from a peripheral location or is naturally excreted or readilyexcreted by said subject.

After capture on the vesicle-capturing material, the vesicles areoptionally washed, and are subsequently used for DNA or RNA extractionfrom the vesicles, and/or identification of proteins or other biomarkerson the vesicle. In several embodiments, multiple aliquots of abiological sample are passed through the device, thereby concentratingthe vesicles from the sample. In some embodiments, the devices andmethods disclosed herein lead to increased sensitivity in severaldiagnostic methods. For example, isolation of vesicles is useful inincreasing sensitivity of diagnostic tests related to vascular disease,as is described in U.S. Provisional Patent Application No. 61/354,117,filed on Jun. 11, 2010, or related to kidney function, as is describedin U.S. Provisional Patent Application No. 61/354,098, filed Jun. 11,2010, the disclosures of each of which are incorporated by referenceherein.

EXAMPLES Example 1 Screening of Filters for the Capacity of ExosomeCollection

One hundred (100) μL of rat plasma was applied to a vesicle-capturedevice as described herein in duplicate, where various vesicle capturematerials (A-K shown in FIG. 1) were inserted at the bottom of eachsample receiving well. The passing through fraction was collected andmixed with 2× Lysis Buffer. The resultant lysate was subject to poly(A)+RNA preparation and cDNA synthesis using an oligo(dT)-immobilizedmicroplate. Further information regarding the oligo(dT)-immobilizedmicroplate can be found in U.S. Pat. No. 7,745,180, issued on Jun. 29,2010, and Mitsuhashi et al., Quantification of mRNA in whole blood byassessing recovery of RNA and efficiency of cDNA synthesis. Clin. Chem.52:634-642, 2006, the disclosure of each of which is incorporated bereference herein. Various mRNAs (group-specific component (gc),apolipoprotein H (apoh), solute carrier organic anion transporter familymember 1b2 (slc1b2), plasminogen (plg), α1-microglobulin/bikuninprecursor (ambp), and albumin (alb)) were then quantified by real timePCR. As a control, an aliquot of the original rat plasma sample was alsomixed with 2× Lysis Buffer and mRNA was quantified simultaneously. Theamount of each gene amplified is shown as 100% (dark horizontal line inFIGS. 1A-1F). The values of mRNA in the passing through fraction (e.g.,the vesicle-depleted sample) were expressed as % Control by using thevalues of original plasma sample. Duplicate experiments measuringabsorption (e.g., capture vesicles by the filters) are shown by the opencircles (o) in FIGS. 1A-1F. The amounts of each of the 6 different mRNAsisolated and amplified from the passing through fraction were reducedwhen plasma was filtered. Lower values for the % Control value for eachfilter tested are representative of a greater degree of capture ofvesicles by the filters (fewer vesicles present in passing throughfraction yields lower generation of PCR product, and hence, a lower %Control). Accordingly, these results indicate that filters A: glassfiber(GF/F), B: nitrocellulose (NC), E: nano alumina fibers, and K: aleukocyte capture membrane perform well at capturing vesicles.

In several embodiments, merely capturing the vesicles from a biologicalsample is insufficient for use in a diagnostic or analytical method.Elution of the vesicles from the filter, extraction of DNA or RNA orprotein from the captured vesicles, or use of the vesicles themselves intests is desirable in some embodiments. To assess the ability toretrieve RNA from the captured vesicles, each of the filters was thentreated with 1× Lysis Buffer, followed by poly(A)+ RNA preparation andcDNA synthesis on oligo(dT)-immobilized microplates, and real time PCR.As shown by the filled triangles (▴) in FIGS. 1A-1F, vesicle-associatedmRNA was detected only from glassfiber (A), nitrocellulose (B), andleukocyte-capture membrane (K). Despite removing vesicles from theplasma, nano alumina fiber-trapped vesicles did not generate any PCRproducts, indicating that, despite lysis of the vesicles, RNA could notbe recovered. Despite showing an absorption capacity similar to otherfilters, glassfiber filters unexpectedly yielded the greatest amount ofassociated PCR product generation (e.g., closest to 100% of Control forall six genes tested).

Example 2 Exosome Capture by Silica Particles

Four mL urine samples from a single donor were applied to avesicle-capture device as described herein (X-axis) or a spin columnpacked with silica particles (Qiagen RNeasy Mini-prep) (Y-axis). Aftercentrifugation at 2,000×g for 5 min, Lysis Buffer was applied andincubated at 55° C. for 30 min. Lysate was then transferred to anoligo(dT)-immobilized microplate for mRNA purification, followed by cDNAsynthesis and real time PCR, according to the established protocols(e.g., those described in U.S. Pat. No. 7,745,180). Experiments wereperformed in triplicate. As shown in FIG. 2, mRNA was detected usingeither glassfibers or silica particles, although glassfibers showedbetter recovery than that of silica particles.

Example 3 Exosome Capture from Saliva

Ten, 1000 μL, saliva samples from a single donor were applied to avesicle-capture device as described comprising glassfiber filters. Aftercentrifugation at 2,000×g for 5 min, Lysis Buffer was applied andincubated at 55° C. for 30 min. Lysate was then transferred to anoligo(dT)-immobilized microplate for mRNA purification, followed by cDNAsynthesis and real time PCR, according to protocol discussed above andfurther described in U.S. Pat. No. 7,745,180. As shown in FIG. 3,various mRNAs (ACTB, B2M, IL8, and trypsin) were detected from saliva.Consequently, the filter-based methods and devices disclosed herein arecapable of capturing exosomes from saliva samples without the need forcostly ultracentrifugation and with decreased risk of contamination (dueto the more streamlined protocol).

Example 4 Visualization of Captured Materials on Glassfiber Filter

Human urine was applied to a glassfiber filter and subsequently washedwith water. Vesicle-laden filters were then analyzed by scanningelectron microscope (SEM). As shown in FIG. 4, small vesicle-likematerials were trapped by the mesh of filters (white arrows in 4A and4B). Vesicles also adhered to the fiber surfaces (white arrows in 4C and4D). Occasionally, the aggregates of vesicles were also trapped by thefilter (white arrows in 4E and 4F). According to the method discussedabove, it was confirmed that this filter at least contained β-actin(ACTB), solute carrier family 12A1 (SLC12A1), and uromodulin (UMOD)mRNA, which are all markers that could be used as diagnostic or controlmarkers from a urine sample. Thus, the devices and methods disclosedherein are well suited for capture of vesicles from urine sampleswithout the need for costly ultracentrifugation and with decreased riskof contamination.

Example 5 Scanning Electron Microscopic Analysis of Exosomes Capturefrom Urine

Urine contains various salts in addition to useful biomarkers. Thesesalts, especially salt crystals, may mask the visualization of exosomesin SEM visualization. Consequently, before undertaking SEM analysis ofexosomes on a filter, in several embodiments, the filter should bewashed with water to eliminate any salts. However, in other embodiments,salts do not interfere, and filter washing is not required. As shown inFIG. 5, various mRNAs (ACTB, ALB, SLC12A1, and UMOD) were quantifiedfrom a glassfiber filterplate with (Y-axis) or without (X-axis) a waterwash. The results indicate that washing with water did not significantlyaffect the detection of mRNA. Consequently, exosomes can be isolatedwith fewer impurities and lower levels of contamination as compared totraditional methods of exosome isolation (e.g., those that requireultracentrifugation, multiple solute transfers to multiple tubes, and/ormultiple rounds of purification). In several embodiments, depending uponthe source of the biological sample, washings with water and additionalfluids is incorporated to further remove impurities and afford enriched,highly pure exosome capture.

SEM analysis was then performed and the low magnification results areprovided in FIG. 6. FIG. 6A shows plain glassfiber before urineapplication, FIG. 6B shows urine application followed by water wash ofthe filter, and FIG. 6C shows urine application followed by lysis bufferapplication to the filter, 55° C. incubation for 30 min, and asubsequent water wash. Bright corner and side edge in FIG. 6A areartifact and were due to charging. The bright areas in B were due tourine application. C was similar to A except for a small area thatappears to be a remnant after lysis and washing. This analysis indicatesthat the filter-based methods and devices afford not only the efficientcapture of exosomes and similar condensed materials, but also allow forthe easy and efficient manipulation of exosomes on the filter. This easeand efficiency is particularly advantageous for sample analyses anddiagnostic methods and is amenable to high-throughput variations ofanalyses and diagnostic methods.

Example 8 Comparison to Ultracentrifugation Method

Various volumes of human urine were applied to glassfiber filters,followed by mRNA quantification as described above. As shown in FIG. 7(Δ), ACTB, UMOD, and SLA12A1 mRNA were quantified in a dose dependentmanner. The maximal urine volume applied to the filter was 3 mL (to asingle well of a 96-well filterplate). In parallel, aliquots of the sameurine aliquots subjected to an art-established ultracentrifugationprotocol (40,000×g for 30 min) to isolate vesicles. The resultantvesicle pellets were dissolved in 1× Lysis buffer, followed by mRNApreparation, cDNA synthesis and real time PCR (). As shown in FIG. 7,the filter-based methods and devices yielded results that were notsignificantly different than the traditional ultracentrifuge method.However, the above discussion illustrates that unlikeultracentrifugation, the filter-based methods and devices are amenableto manipulation during sample isolation or enrichment. Thesemanipulations include, but are not limited to water washing, bufferwashing, lysis on the filter, etc. Consequently, the filter-basedmethods represent a highly versatile and easy to use alternative toultracentrifugation that does not sacrifice sensitivity in the detectionof the desired biomarker.

Example 9 Determination of Zeta Potential of Vesicle Capture Materials

The zeta potential of a material is one measure of the ability thatmaterial to attract particles. Based on the disclosure above, theability to attract (e.g., filter) vesicles from a biological fluid isbeneficial in analysis of vesicle-associated biomarkers. While theoverall surface charge on a vesicle will depend on the lipid and proteinmake-up of the vesicle, as well as the pH of the fluid in which thevesicle is contained, the vesicle capture media can be chosen (and ormodified) in order to provide improved attractive forces to thevesicles. As shown in FIGS. 8A-8B, the zeta potential for polystyrenevesicle capture material was calculated as negative 5.72 mV. FIGS.9A-9B, depict the zeta potential for PVDF Immobilon-P vesicle capturematerial, which was calculated as negative 1.71 mV. FIGS. 10A-10B depictthe zeta potential for glassfiber, which was calculated as negative 2.60mV. Finally, FIGS. 11A-11B depict the zeta potential for mylon nytran,which was calculated as positive 1.03 mV. As also shown in FIGS. 8B, 9B,10B, and 11B, each vesicle capture material showed a varied strength ofattraction (y-axis), which is representative of the ability of thematerial to hold the vesicle. These data suggest that, depending on thecharge of the vesicles to be captured, certain materials (orcombinations of materials) may be optimally suited for a certain vesicletype. For example, if a target vesicle is positively charged, apolystyrene vesicle capture material may provide the greatest attractiveforces to capture the vesicle from the fluid. As discussed above,however, release of the vesicle from the vesicle capture material isalso important in some embodiments. Thus, the attractive forces (thezeta potential), in several embodiments, are balanced with the strengthof attraction (negatively correlated with the release of vesicles fromthe material). In some embodiments, the vesicle capture materials may bemodified (as discussed above) in order to tailor their vesicle-capturingprofile.

1. A method of isolating vesicles from biological fluid, comprising: (a)obtaining a biological fluid sample comprising said vesicles; (b)loading at least a portion of said biological fluid sample into a sampleloading region of a vesicle capture device; (c) passing said biologicalfluid sample from said sample loading region through a vesicle-capturematerial in said vesicle capture device, said vesicle-capture materialcomprising at least a first layer and a second layer of glassfiber,thereby producing a supernatant; and (d) passing said supernatant to asample receiving region of said vesicle capture device and discardingthe supernatant, wherein said passings result in capture of saidvesicles from said biological fluid sample on or in said vesicle-capturematerial.
 2. The method of claim 1 wherein said vesicle-capture materialcaptures vesicles sized from about 0.6 microns to about 1.6 microns indiameter.
 3. The method of claim 1, wherein said vesicle-capturematerial captures exosomes or other vesicles ranging in size from about0.020 to about 1.0 microns.
 4. (canceled)
 5. (canceled)
 6. The method ofclaim 1, wherein step (c) comprises passing said biological fluidthrough said first layer of glassfiber so as to capture material fromsaid biological sample that is about 1.6 microns or greater in diameter.7. The method of claim 6, wherein step (c) further comprises passingsaid biological fluid through said second layer of glassfiber so as tocapture vesicles having a minimum size from about 0.6 microns to about0.8 microns in diameter, and having a maximum size of less than 1.6microns.
 8. The method of claim 1, wherein said vesicle-capture materialfurther comprises a non glass-like material.
 9. The method of claim 8,wherein said non glass-like material comprises nitrocellulose.
 10. Themethod of claim 1, wherein said passings are accomplished through theapplication of vacuum pressure to the device.
 11. The method of claim 1,wherein said passings are accomplished through the application ofpositive pressure to the device.
 12. The method of claim 1, wherein saidpassings are accomplished through low-speed centrifugation of thedevice.
 13. The method of claim 1, wherein said vesicle capture deviceis configured in a multi-well plate format.
 14. The method of claim 1,further comprising eluting said vesicles from said vesicle-capturematerial.
 15. A method of isolating a biomarker, comprising: capturingvesicles from a biological fluid sample according to claim 1, whereinsaid vesicles comprise at least one biomarker; removing non-vesiclematerial from said device; and lysing said captured vesicles in or onsaid vesicle-capture material with a lysis buffer, thereby isolating abiomarker from said vesicles.
 16. The method of claim 15, wherein saidbiomarker is selected from the group consisting of RNA, DNA, protein,and carbohydrate.
 17. The method of claim 16, wherein said RNA is of atype selected from the group consisting of mRNA, miRNA, rRNA, tRNA, andvRNA. 18.-22. (canceled)